Patients with irritable bowel syndrome (IBS) suffer from chronic abdominal pain and associated GI symptoms - diarrhea, constipation, bloating & urgency. Those with severe symptoms may also have visceral hypersensitivity to nociceptive stimuli, suggesting aberrant neural pain processing mechanisms. Indeed, we found a significant subset (~35%) of IBS patients with visceral hypersensitivity to noxious experimental stimuli (e.g., colonic distension); but the mechanism was unclear. We found a clue to that mechanism during our previous NIH-supported study (R01NS053090); we identified unique microRNAs (miRNAs) in colon tissues of this subset. Here we will follow up on that clue by testing the following hypothesis: Aberrant expression of key miRNAs dysregulates expression of downstream miRNA targets ( opioid receptors; NMDA receptors) which contributes to visceral hypersensitivity in IBS patients. To determine the intra- and inter-cellular roles of unique miRNAs in the epigenetic regulation of down- stream receptor target genes, our lab has developed innovative methods. These methods combine unique miRNA recognition properties of locked nucleic acid (LNA) modified oligonucleotide probes with fluorescence in situ hybridization (FISH) using tyramide signal amplification (TSA) technology. We will use in vivo intrathecal injection of miRNA inhibitors (LNAs) or miRNA mimics (precursors) to experimentally regulate expression of specific miRNAs that target opioid (Aim 1) and glutamate (Aim 2) receptors in our validated rat model of visceral hypersensitivity (Zhou et al., Pain, 2008). Thus, we will use a combination of strategies to develop a comprehensive high-confidence model of miRNA targeting networks for opioid and glutamine receptors. In Aim 1 we will determine whether increased miR-29a/b cluster and miR-29b-1-5p expression enhances visceral hypersensitivity via downregulation of opioid signaling pathways. In Aim 2, we will determine whether decreased miR-23a/b cluster and 125b-1-3p expression facilitates visceral hypersensitivity via upregulation of glutamate signaling mechanisms. Our proposed studies should identify critical contributions of aberrantly expressed miRNAs that target downstream opioid and glutamate receptors in intestinal tissues of IBS patients with visceral hypersensitivity This will bridge an important gap in our knowledge of the underlying mechanisms of dysregulation of nociceptive pathways that lead to chronic pain and altered visceral nociception in patients who suffer from common functional GI disorders. More importantly, the results should lead to the development of innovative therapeutic strategies via development of synthesized oligonucleotides that inhibit or mimic specific miRNAs that modulate epigenetic regulation of down-stream target genes (e.g. opioid or glutamate receptors) that drive visceral nociception. The ultimate goal of the proposed translational studies is to develop new therapies for treating our patients with IBS and other functional GI disorders.